The direct injection of DNA into the pronuclei of fertilized mouse eggs has been used to produce transgenic mice (See, e.g., U.S. Pat. No. 5,557,032 and U.S. Pat. No. 5,175,383). Microinjection has also been used to produce other transgenic animals including, for example, cows (Haskell et al., Mol. Reprod. Dev. (United States), 40(3): 386-390 (1995)), sheep (Rexroad et al., Mol. Reprod. Dev. (United States), 1(3): 164-169 (1989)), and pigs (Pursel et al., Vet. Immunol. Immunopathol. (Netherlands), 17(1-4): 303-313 (1987)). Although the frequency of non-murine, transgenic animals obtained by this method is quite low, some transgenic ungulates have purportedly been shown to transmit the transgene to their progeny (See, e.g., Rexroad et al., Id. and Pursel et al., Id.). However, the direct injection of DNA into pronuclei does not provide an opportunity to manipulate or otherwise control DNA integration.
In contrast to the pronuclei direct injection technique, DNA integration can be controlled by introducing the DNA into embryonic stem (ES) cells and then injecting the transfected cells into embryos, where they become incorporated into the developing embryos. Manipulation of ES cells by this method has been used to produce transgenic mice which contain transgenes that, for example, decrease or completely suppress the expression of endogenous genes (U.S. Pat. No. 5,557,032); encode human immunoglobulin genes (U.S. Pat. No. 5,569,825); and, encode the tumor suppressing p53 gene (U.S. Pat. No. 5,569,824).
In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei from mouse embryos which beyond the eight-cell stage after transfer reportedly do not support the development of enucleated oocytes (Cheong et al, Biol. Reprod 48:958 (1993)). Therefore, ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures. However, to date, the production of transgenic ungulate embryos produced from transgenic ES cells has not been reported.
Methods for deriving ES cell lines in vitro from early preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol., 121:1-9 (1987)) is present.
ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early development. Mouse ES cells can give rise to germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309:255-256 (1984)).
Many research groups have reported the isolation of purportedly pluripotent embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43:255-260 (1991), report the establishment of purportedly stable, pluripotent cell lines from pig and sheep blastocysts which exhibit some morphological and growth characteristics similar to that of cells in primary cultures of inner cell masses isolated immunosurgically from sheep blastocysts. (Id.) Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:51-56 (1990) discloses maintenance and differentiation in culture of putative pluripotential embryonic cell lines from pig blastocysts. Further, Gerfen et al., Anim. Biotech, 6(1): 1-14 (1995) disclose the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned medium. These cells reportedly differentiate into several different cell types during culture (Gerfen et al., Id.).
Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992) report bovine embryonic stem cell-like cell lines which, when cultured, survived passages for three, but were lost after the fourth passage. Still further, Handyside et al., Roux's Arch. Dev. Biol., 196:185-190 (1987) disclose culturing of immunosurgically isolated inner cell masses of sheep embryos under conditions which allow for the isolation of mouse ES cell lines derived from mouse ICMs. Handyside et al. (1987) (Id.), report that under such conditions, the sheep ICMs attach, spread, and develop areas of both ES cell-like and endoderm-like cells, but that after prolonged culture only endoderm-like cells are evident. (Id.)
Recently, Cherny et al., Theriogenology, 41:175 (1994) reported purportedly pluripotent bovine primordial germ cell-derived cell lines maintained in long-term culture. These cells, after approximately seven days in culture, produce ES-like colonies which stain positive for alkaline phosphatase (AP), exhibit the ability to form embryoid bodies, and spontaneously differentiate into at least two different cell types. These cells also reportedly express mRNA for the transcription factors OCT4, OCT6 and HES1, a pattern of homeobox genes which is believed to be expressed by ES cells exclusively. In addition, First et al., Reprod. Fertil. Dev. (Australia), 6(5): 553-62 (1994) also report the establishment of bovine embryonic cell cultures from blastocyst ICM cells which "exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture" (see Abstract). However, these researchers also report that "The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined." (Id.).
Also recently, Campbell et al., Theriogenology, 43:181 (1995) in an abstract reported the production of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day nine ovine embryos cultured under conditions which promote the isolation of ES cell lines in the mouse. The authors conclude based on their results that ED cells from day nine ovine embryos are totipotent by nuclear transfer and that totipotency is maintained in culture for up to three passages.
Even more recently, Campbell et al, Nature, 380:64-68 (1996) reported cloning of sheep by nucleic transfer from a cultured cell line. However, the cells used are dissimilar to the CICM's of the present invention. Unlike the subject CICM cells, the cells of Campbell et al formed a monolayer in tissue culture. The authors refer to these cells as being "flattened" or as exhibiting an "epithelial" appearance. By contrast, the CICM cells of the present invention can be continually maintained in a multilayer colony or portions of the colony when grown in an undifferentiated state. Also, the cells of Campbell et al are cytokeratin and laminin A/C positive. By contrast, the CICM cells of the present invention are cytokeratin negative.
Moreover, there is no suggestion that the cells of Campbell et al are undifferentiated. Rather, the reference only indicates that these cells are useful in nucleic transfer procedures. Also, these cells are not cultured under conditions wherein they maintain constant contact with a fibroblast feeder layer. Rather, the cultured cells (of Campbell et al (1996)) apparently push the fibroblasts to the side in culture and grow on top of the culture dish.
Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported the isolation and characterization of purportedly permanent cell lines from inner cell mass cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or 9 day bovine blastocysts under different conditions to determine which feeder cells and culture media are most efficient in supporting the attachment and outgrowth of bovine ICM cells. They concluded based on their results that the attachment and outgrowth of cultured ICM cells is enhanced by the use of STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial cells) and by the use of charcoal-stripped serum (rather than normal serum) to supplement the culture medium. Van Stekelenburg et al report, however, that their cell lines resembled epithelial cells more than pluripotent ICM cells. (Id.)
Still further, Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et al, WO 90/03432, published Apr. 5, 1990 and Wheeler et al, WO 94/26889 published Nov. 24, 1994 report the isolation, selection and propagation of animal stem cells which purportedly may be used to obtain transgenic animals. Also, Evans et al., WO 90/03432, published on Apr. 5, 1990, report the derivation of purportedly pluripotent embryonic stem cells derived from porcine and bovine species which are asserted to be are useful for the production of transgenic animals. Further, Wheeler et al., WO 94/26884, published Nov. 24, 1994 and U.S. Pat. No. 5,523, 226, issued Jun. 4, 1996, disclose embryonic stem cells which are asserted to be useful for the manufacture of chimeric and transgenic ungulates. The method disclosed by Wheeler differs from the instant claimed invention in that the Wheeler method requires that the dissociated cells from swine embryos be cultured in "conditioned stem cell medium" and that the resultant subcultured cells then be introduced into a SCID mouse. Thus, based on the foregoing, it is evident that many groups have attempted to produce ES cell lines, e.g., because of their potential application in the production of cloned or transgenic embryos and in nuclear transplantation.
The use of ungulate ICM cells for nuclear transplantation has also been reported. For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994) disclose nuclear transplantation of bovine ICMs by microinjection of the lysed donor cells into enucleated mature oocytes. The reference discloses culturing of embryos in vitro for seven days to produce fifteen blastocysts which, upon transferral into bovine recipients, resulted in four pregnancies and two births. Also, Keefer et al., Biol. Reprod., 50:935-939 (1994), disclose the use of bovine ICM cells as donor nuclei in nuclear transfer procedures, to produce blastocysts which, upon transplantation into bovine recipients, resulted in several live offspring. Further, Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993), disclose the production of calves by transfer of nuclei from short-term in vitro cultured bovine ICM cells into enucleated mature oocytes.
Also, the production of live lambs following nuclear transfer of short-term cultured embryonic disc cells (up to three passages) has been reported (Campbell et al., Theriogenology, 43:181 (1995)). Still further, the use of bovine pluripotent embryonic cells in nuclear transfer and the production of chimeric fetuses has also been reported (Stice et al., Theriogenology, 41:301 (1994)).
However, notwithstanding what has been previously reported in the literature, there still exists a significant need for cultured ICM cells and cell lines which possess improved properties, e.g., which possess morphological properties and express cell markers identically or substantially similar to ICM cells of developing embryos, in particular ungulate embryos. There further exists a significant need in the art for methods of producing such improved cultured ICM cells and cell lines. In addition, there exists a significant need for reliable, efficient methods to manipulate or otherwise control heterologous DNA integration into non-human embryos in order to produce non-human transgenic embryos, fetuses, and whole animals. There further exists a significant need in the art for methods of producing non-human, non-murine transgenic fetuses whose germ line cells contain heterologous DNA. Transgenic whole animals produced from such transgenic fetuses can transmit the heterologous DNA to their progeny thereby fulfilling a long-felt need for reliable, efficient methods of producing non-human, non-murine transgenic animal progeny.